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Molecular diagnostic point-of-care (MDx POC) testing is gaining momentum and is increasingly important for infectious disease detection and monitoring, as well as other diagnostic areas such as oncology. Molecular testing has traditionally required high-complexity laboratories. Laboratory testing complexity is determined by utilizing the Clinical Laboratory Improvement Amendments of 1988 (CLIA) Categorization Criteria scorecard, utilizing seven criteria that are scored on a scale of one to three. Previously, most commercially available point-of-care (POC) tests use other analytes and technologies that were not found to be highly complex by the CLIA scoring system. However, during the COVID-19 pandemic, MDx POC testing became much more prominent. Utilization during the COVID-19 pandemic has demonstrated that MDx POC testing applications can have outstanding advantages compared to available non-molecular POC diagnostic tests. This article introduces MDx POC testing to students, technologists, researchers, and others, providing a general algorithm for MDx POC test development. This algorithm is an introductory, step-by-step decision tree for defining a molecular POC diagnostic device meeting the functional requirements for a desired application. The technical considerations driving the decision-making include nucleic acid selection method (DNA, RNA), extraction methods, sample preparation, number of targets, amplification technology, and detection method. The scope of this article includes neither higher-order multiplexing, nor quantitative molecular analysis. This article covers key application considerations, such as sensitivity, specificity, turnaround time, and shipping/storage requirements. This article provides an overall understanding of the best resources and practices to use when developing a MDx POC assay that may be a helpful resource for readers without extensive molecular testing experience as well as for those who are already familiar with molecular testing who want to increase MDx availability at the POC. © 2024 Wiley Periodicals LLC., (© 2024 Wiley Periodicals LLC.)

Myotonic dystrophy (DM), the most common inherited muscle disorder, is caused by a CTG expansion in the 3"-untranslated region of a protein kinase gene ( DMPK ). The complex and variable phenotype is most likely caused by a complex molecular pathogenesis, including deficiency of the DMPK protein, a trans -dominant misregulation of RNA homeostasis and haploinsufficiency of a neighboring homeobox gene [DM locus-associated homeodomain protein (DMAHP )]. Here, we study the allele-specific transcriptional activity of the DMAHP/SIX5 gene in DM patient tissues. Using a quantitative fluorescent RT-PCR assay, we tested allele-specific accumulation of DMAHP/SIX5 transcripts in both total and poly(A)+pools. In muscle biopsies, we found that transcript reductions of DMAHP/SIX5 alleles in cis with CTG expansions correlated with the extent of expansion. A patient with approximately 90 CTG repeats in muscle DNA (normal n < 37) showed a 20% reduction of allele-specific transcript levels, while four other DM patients with larger expansions showed 80% reductions. The effects of the CTG expansions on DMAHP transcription were tissue specific: autopsy tissues from a patient with 1500 repeats showed 80% reductions in muscle and liver; however, RNA from other tissues (lung, aorta, heart conduction tissue, cerebellum) showed 0-20% reductions. Our results suggest that the effect of the CTG repeat on the DMAHP/SIX5 promoter is variable and tissue-specific. Our data are consistent with abnormalities of DMAHP/SIX5 probably having a more prominent role in disease pathogenesis in muscle, liver and brain, but being less important in other tissues.